Devices and methods for capturing target molecules

ABSTRACT

Provided herein are devices and methods for the capture or isolation of a biomarker from a biological sample. In several embodiments, the device comprises a loading region, a filter material, and a receiving region. In particular, in several embodiments, biological fluid is passed from the loading region through the filter material and into the receiving region, thereby resulting in capture or isolation of a biomarker.

RELATED CASES

The entire disclosure of each of the applications listed in theaccompanying Application Data Sheet is incorporated by reference herein.

BACKGROUND

Field

The present disclosure relates to systems, devices and methods for theenhanced efficiency of capturing agents of interest from fluid samples.The fluid samples are biological fluid samples in some embodiments,while in other embodiments, non-biological fluid samples are used. Forexample, in several embodiments, environmental water samples are passedthrough the devices as disclosed herein in order to assess, for example,mineral content, pollution levels, chemical or toxin content, presenceof pathogens, etc.

Description of Related Art

Often it is desirable to extract certain components from a liquid. Forexample, many medical tests analyze biomarkers in a fluid sample (e.g.,blood, urine, etc.) taken from a patient. Diagnosis or prognosis may bederived from identification of a biomarker or a biochemical pattern thatis not present in healthy patients or is altered from a previouslyobtained patient sample.

Frequently, the use of bodily fluids to isolate or detect a biomarkersignificantly dilutes the biomarker. Moreover, most biomarkers areproduced in low or even moderate amounts in tissues and bodily fluids.Diagnosis or prognosis is likely less accurate when the compounds ofinterest are present at low concentrations.

SUMMARY

In several embodiments, there are provided systems and methods for theefficient collection of biomarkers from fluid samples, such asbiological fluid samples. For example, in several embodiments, there isprovided a system for capturing vesicles from a biological fluid sampleobtained from a subject, comprising (i) a vesicle capture device,comprising (a) a first body having an inlet, an outlet, and an interiorvolume between the inlet and the outlet, (b) a second body having aninlet, an outlet, an interior volume between the inlet and the outlet, afilter material positioned within the interior volume of the secondbody, and in fluid communication with said first body, wherein the firstbody and the second body are reversibly connected by an interaction ofthe inlet of the second body with the outlet of the first body, (ii) areceiving vessel having an interior cavity, and wherein the interiorcavity of the receiving vessel is dimensioned to reversibly enclose boththe first and the second body and to receive the biological fluid sampleafter it is passed from the interior volume of the first body, throughthe filter material, through the interior cavity of the second body andout of the outlet of the second body.

In several embodiments, the system further comprises (iii) one or moreanalysis wells configured to reversibly interact with the outlet of anindividual second body. In some embodiments, the analysis wells comprisea single tube, such as, for example, a microcentrifuge tube. In severalembodiments, however, the one or more analysis wells comprise a standard6-, 12-, 48-, or 96-well microplate. In one embodiment, the one or moreanalysis wells comprises a standard 96-well microplate.

Additionally, in several embodiments, there is provided a system forcapturing vesicles from a biological fluid sample obtained from asubject, comprising (i) a vesicle capture device, comprising (a) a firstbody having an inlet, an outlet, and an interior volume between theinlet and the outlet, (b) a second body having an inlet, an outlet, aninterior volume between the inlet and the outlet, a filter materialpositioned within the interior volume of the second body, and in fluidcommunication with said first body, wherein the first body and thesecond body are reversibly connected by an interaction of the inlet ofthe second body with the outlet of the first body, wherein saidinteraction comprises insertion of the outlet of the first body into theinlet of the second body, wherein the second body is disconnected fromthe first body by extracting the outlet of the first body from the inletof the second body, wherein the outlet of the second body is dimensionedto fit within the inner diameter of a well of a 6, 12, 48, or 96-wellmicroplate, wherein the second body comprises a unique identifier thatcorresponds to the identity of said subject; and (ii) a receiving vesselhaving an inlet, a closed end opposite the inlet and interior cavity,wherein the interior cavity of the receiving vessel is dimensioned toreversibly enclose both the first and the second body and to receive thebiological fluid sample after it is passed from the interior volume ofthe first body, through the filter material, through the interior cavityof the second body and out of the outlet of the second body.

In several embodiments, the interior volume of the first body rangesfrom about 2 to about 10 mL. In several embodiments, the interior volumeof the second body ranges from about 2 to about 5 mL.

In several embodiments, the reversible connection between the inlet ofthe second body with the outlet of the first body comprises a frictionconnection. In several embodiments, the reversible connection betweenthe inlet of the second body with the outlet of the first body comprisesa luer lock connection. In several embodiments, the reversibleconnection between the inlet of the second body with the outlet of thefirst body comprises rotational connection wherein a pin on the outletof the second body mates with a groove on the inlet of the second body.In several embodiments, the reversible connection between the inlet ofthe second body with the outlet of the first body comprises a threadedconnection. In some embodiments, the inlet of the second body comprisesfemale threads on the inlet of the second body that mate with malethreads on the outlet of the first body. In additional embodiments, theinlet of the second body comprises male threads on the inlet of thesecond body that mate with female threads on the outlet of the firstbody. In several embodiments, the connection is configured to allow thesecond body to be coupled and later de-coupled from the first bodywithout the second body having to pass through the interior volume ofthe first body.

In several embodiments, the unique identifier on the second bodycomprises a patient specific RFID tag. In several embodiments, theunique identifier on the second body comprises a patient specific2-dimensional bar code. In several embodiments, the unique identifier onthe second body comprises a patient specific 3-dimensional bar code. Inseveral embodiments, the unique identifier on the second body is visibleand/or readable from a position above the second body, when the outletof said second body is in communication with analysis wells. In severalembodiments, other visual, electronic, or magnetic unique patientidentifiers are used.

In several embodiments, the interior cavity of the receiving vessel hasa volume of about 50 mL. In several embodiments, the interior cavity ofthe receiving vessel has a volume of about 100 mL.

In several embodiments, the first body comprises a lip extending from aperimeter of the inlet of the first body, and wherein said lip rests ona perimeter of the inlet of the receiving vessel and allows thereceiving vessel to enclose the first and second bodies while allowingremoval of the first and second bodies from the receiving vessel. Inseveral embodiments, the lip on the first body holds the first andsecond body in a fixed position within the interior cavity of thereceiving vessel. Advantageously, in several embodiments, the fixedposition is a position in which the outlet of the second body does notcontact the biological fluid sample that has passed from the interiorvolume of the first body, through the filter material and into theinterior cavity of the receiving vessel.

In several embodiments, the receiving vessel consists essentially of theinlet, the closed end opposite the inlet and the interior cavity (e.g.,the receiving vessel has no additional ports or openings).

In several embodiments, centrifugation is used to pass said biologicalfluid sample from the interior of the first body, through the filtermaterial, out the outlet of the second body and into the interior volumeof the receiving vessel.

In several embodiments, the filter material comprises a plurality oflayers of one or more glass-like materials configured to retain vesicleshaving a diameter of from about 0.6 microns to about 1.5 microns indiameter.

In several embodiments, the system does not employ negative or positivepressure to pass said biological fluid sample from the interior of thefirst body, through the filter material, out the outlet of the secondbody and into the interior volume of the receiving vessel.

There are additionally provided methods for isolating vesicles from abiological fluid sample, the methods comprising, obtaining a systemcomprising a first body having an inlet, an outlet, and an interiorvolume between the inlet and the outlet, a second body having an inlet,an outlet, an interior volume between the inlet and the outlet, a filtermaterial positioned within the interior volume of the second body, andin fluid communication with said first body, wherein the first body andthe second body are reversibly connected by an interaction of the inletof the second body with the outlet of the first body, a receiving vesselhaving an interior cavity, and wherein the interior cavity of thereceiving vessel is dimensioned to reversibly enclose both the first andthe second body and to receive the biological fluid sample after it ispassed from the interior volume of the first body, through the filtermaterial, through the interior cavity of the second body and out of theoutlet of the second body, placing a volume of a biological fluid sampleinto interior volume of the first body via the inlet of the first body,wherein the first body is reversibly connected to the second body,placing the first and second bodies into the interior cavity of thereceiving vessel, applying one or more of gravitational or centrifugalforce to the receiving vessel, thereby causing the biological sample topass from the interior volume of the first body, through the filtermaterial of the second body, out the outlet of the second body and intothe interior cavity of the receiving vessel, wherein the filter capturesvesicles from the biological fluid sample, thereby isolating a vesiclefrom said biological fluid sample.

In several embodiments, the method further comprises detectingexpression of a gene of interest from biological fluid sample,extracting the first and second bodies from the receiving vessel;disconnecting the first and second body from one another; placing theoutlet of the second body in communication with a well of a 96-wellmicroplate; introducing an elution buffer into the inlet of the secondbody, wherein said elution buffer comprises a buffer that lyses saidvesicles captured on the filter of the second body, thereby releasingRNA from said vesicle; transferring said released RNA from said filterto the corresponding well of the 96-well microplate; and detectingexpression of said gene of interest by a method comprising: (i)contacting said RNA with a reverse transcriptase to generatecomplementary DNA (cDNA), and (ii) contacting said cDNA with sense andantisense primers that are specific for said gene of interest and a DNApolymerase to generate amplified DNA, thereby detecting expression ofsaid gene of interest. In additional embodiments, other microplate sizes(or individual tubes or arrays of tubes) may also be used.

Additionally provided is a system for capturing vesicles from abiological fluid sample obtained from a subject, comprising (i) avesicle capture device, comprising: (a) a first body having an inlet, anoutlet, and an interior volume between the inlet and the outlet; (b) asecond body having an inlet, an outlet, an interior volume between theinlet and the outlet, a filter material positioned within the interiorvolume of the second body, having a unique identifier comprising apatient specific RFID tag, and in fluid communication with said firstbody, wherein the first body and the second body are reversiblyconnected by an interaction of the inlet of the second body with theoutlet of the first body; (ii) a receiving vessel having an interiorcavity, and wherein the interior cavity of the receiving vessel isdimensioned to reversibly enclose both the first and the second body andto receive the biological fluid sample after it is passed from theinterior volume of the first body, through the filter material, throughthe interior cavity of the second body and out of the outlet of thesecond body.

Additionally provided is a system for capturing vesicles from abiological fluid sample obtained from a subject, comprising: (i) avesicle capture device, comprising: (a) a first body having an inlet, anoutlet, and an interior volume between the inlet and the outlet; (b) asecond body having an inlet, an outlet, an interior volume between theinlet and the outlet, a filter material positioned within the interiorvolume of the second body, having a unique identifier comprising apatient specific 2-dimensional bar code, and in fluid communication withsaid first body, wherein the first body and the second body arereversibly connected by an interaction of the inlet of the second bodywith the outlet of the first body; (ii) a receiving vessel having aninterior cavity, and wherein the interior cavity of the receiving vesselis dimensioned to reversibly enclose both the first and the second bodyand to receive the biological fluid sample after it is passed from theinterior volume of the first body, through the filter material, throughthe interior cavity of the second body and out of the outlet of thesecond body.

Additionally provided is a system for capturing vesicles from abiological fluid sample obtained from a subject, comprising: (i) avesicle capture device, comprising: (a) a first body having an inlet, anoutlet, and an interior volume between the inlet and the outlet; (b) asecond body having an inlet, an outlet, an interior volume between theinlet and the outlet, a filter material positioned within the interiorvolume of the second body, having a unique identifier comprising apatient specific 3-dimensional bar code, and in fluid communication withsaid first body, wherein the first body and the second body arereversibly connected by an interaction of the inlet of the second bodywith the outlet of the first body; (ii) a receiving vessel having aninterior cavity, and wherein the interior cavity of the receiving vesselis dimensioned to reversibly enclose both the first and the second bodyand to receive the biological fluid sample after it is passed from theinterior volume of the first body, through the filter material, throughthe interior cavity of the second body and out of the outlet of thesecond body.

Additionally provided is a system for capturing vesicles from abiological fluid sample obtained from a subject, comprising: (i) avesicle capture device, comprising: (a) a first body having an inlet, anoutlet, and an interior volume between the inlet and the outlet; (b) asecond body having an inlet, an outlet, an interior volume between theinlet and the outlet, a filter material positioned within the interiorvolume of the second body, and in fluid communication with said firstbody, wherein the first body and the second body are reversiblyconnected by an interaction of the inlet of the second body with theoutlet of the first body, wherein said interaction comprises aninteraction between female threads on the inlet of the second body thatmate with male threads on the outlet of the first body; (ii) a receivingvessel having an interior cavity, and wherein the interior cavity of thereceiving vessel is dimensioned to reversibly enclose both the first andthe second body and to receive the biological fluid sample after it ispassed from the interior volume of the first body, through the filtermaterial, through the interior cavity of the second body and out of theoutlet of the second body.

Additionally provided is a system for capturing vesicles from abiological fluid sample obtained from a subject, comprising: (i) avesicle capture device, comprising: (a) a first body having an inlet, anoutlet, and an interior volume between the inlet and the outlet; (b) asecond body having an inlet, an outlet, an interior volume between theinlet and the outlet, a filter material positioned within the interiorvolume of the second body, and in fluid communication with said firstbody, wherein the first body and the second body are reversiblyconnected by an interaction of the inlet of the second body with theoutlet of the first body, wherein said interaction comprises aninteraction between male threads on the inlet of the second body thatmate with female threads on the outlet of the first body; (ii) areceiving vessel having an interior cavity, and wherein the interiorcavity of the receiving vessel is dimensioned to reversibly enclose boththe first and the second body and to receive the biological fluid sampleafter it is passed from the interior volume of the first body, throughthe filter material, through the interior cavity of the second body andout of the outlet of the second body.

In several embodiments, the reversible connections between the first andsecond body is configured to allow disconnection of the second body fromthe first body without the second body passing through the interiorvolume of the first body.

In several embodiments, these systems further comprise (iii) one or moreanalysis wells configured to reversibly interact with the outlet of anindividual second body.

In several embodiments wherein a microplate is used, a frame ispositioned between the vesicle capture device second body in order toimprove the stability of the interaction between the second hollow bodyand the microplate. In several embodiments, after placing the secondhollow body into the microplate (e.g., through the frame) a lysis bufferis added and incubated at 37° C. for 1-20 (e.g., 10) minutes to releasemRNA from captured exosomes. In several embodiments, the frame was thenplaced onto oligo(dT)-immobilized plate and centrifuged for 1-10 (e.g.,5) minutes at ˜2000×g at ˜4° C. The resultant oligo(dT)-immobilizedplate was stored at ˜4° C. overnight (e.g., 14 to 24 hours) for thehybridization between poly(A)+ tail of mRNA and immobilized oligo(dT) asdescribed previously. In several embodiments, non-mRNA materials areremoved by washing and cDNA is generated, which can used for real timePCR (or other analysis methods). This process can also be used when anon-microplate format (e.g., tube or array of tubes) is used, as well aswhen no microplate frame is used. Moreover, additional embodiments,employ different reaction conditions and/or times.

Given that accurate diagnosis may be hampered (or even impossible) whena target compound of interest is present in a biological sample at lowconcentrations, there is a need for devices and methods for extractingbiomarkers and other components of interest from a fluid sample of apatient without unduly lowering the concentration of the targetbiomarker. Extraction of fluid components is beneficial in many contextsincluding, but not limited to, filtration, purification, isolation, andenrichment.

Thus, several embodiments of the devices and methods allow extraction oftarget components from liquids. In particular, the devices and methodsdisclosed herein are useful for capturing from biological fluids nucleicacids, exosomes, vesicles, and other circulating membrane bound nucleicacid and/or protein-containing structures. However, as the devices andmethods disclosed herein permit extraction of organic and non-organiccompounds, the devices and methods disclosed herein are applicable tofluid samples of biological or non-biological origin.

Conventional methods of vesicle and exosome extraction often involveultracentrifugation in order to separate the vesicles from other matterin a biological sample. Ultracentrifugation is accomplished through theuse of expensive and potentially hazardous equipment. Moreover,ultracentrifugation often results in samples being collected in multipletubes. Consequently, ultracentrifugation is sometimes an impractical orimpossible technique for many laboratories.

Therefore, provided herein are devices and methods for capture ofnucleic acids, exosomes, vesicles, and other circulating membrane-boundnucleic acid and/or protein-containing structures that are released fromcells into biological fluids. In several embodiments the devices andmethods as disclosed herein provide several advantages over traditionaltechniques for vesicle isolation, such as ultracentrifugation. Forexample, in some embodiments the devices and methods disclosed hereincapture vesicles, exosomes, and/or biomarkers from samples andadvantageously allow multiple filtrations of samples through the samefilter. Consequently, increased amounts of vesicle material can becollected simply by applying multiple sample aliquots to a device. Insome embodiments, vesicle yield is increased by re-passing the filtrateof a sample aliquot through the device.

In several embodiments, there is provided a method of isolating vesiclesfrom biological fluid, comprising: (a) obtaining a biological fluidsample comprising said vesicles; (b) adjusting the salt concentration ofthe fluid sample to between about 10 mM and 800 mM; (c) adjusting the pHof the fluid sample to between about pH 6 and pH 9; and (d) passing saidbiological fluid sample through a vesicle-capture material, saidvesicle-capture material comprising glass-like materials, wherein thevesicles from said biological fluid sample are captured on or in saidvesicle-capture material.

In several embodiments, a sample aliquot is subjected to centrifugationbefore passing the sample aliquot through the vesicle-capture material.In some embodiments, the supernatant generated by said centrifugation isdiscarded, while in other embodiments it is passed through said capturematerials one or more additional times to increase the yield ofvesicles. In several embodiments, the fluid sample is urine. In severalembodiments, the salt concentration is adjusted to between about 20 mMand 600 mM. In several embodiments, the salt concentration is based onthe concentration of monovalent cations in the sample.

In several embodiments, the vesicle-capture material is pre-treated toremove a material that inhibits capture of the vesicles. In severalembodiments, albumin is removed by pretreatment. In several embodiments,the material is pre-treated by a method selected from the groupconsisting of heating, acid bath, basic bath, and ultrasonic cleaning.

In several embodiments, the adjusting comprises titrating said fluidsample with a concentrated salt and buffer solution to reach said saltconcentration of between about 10 mM and 800 mM and said pH of betweenabout pH 6 and about pH 9.

In several embodiments, the vesicle-capture material comprisesglass-like materials. In several embodiments the vesicle-capturematerial comprises a plurality of layers of the material. In someembodiments, the retention rate of the vesicle-capture material isgreater than 50%, 75%, 90% or 99% for vesicles having a diameter of fromabout 0.6 microns to about 1.5 microns in diameter. In one embodiment,the vesicle-capture material captures vesicles sized from about 0.7microns to about 1.6 microns in diameter. In one embodiment, thevesicle-capture material captures exosomes or other vesicles ranging insize from about 0.020 to about 1.0 microns.

In several embodiments, the vesicle-capture material comprises aplurality of layers of material. In several embodiments, combinations ofvesicle capture materials are used. In some embodiments, a plurality ofglass-like materials is used. In several embodiments, the plurality oflayers of the vesicle-capture material comprises at least a first layerand a second layer of glass fiber. In some embodiments, the biologicalfluid is passed through a first layer of glass fiber to capture materialfrom the biological sample that is about 1.6 microns or greater indiameter. In some embodiments, the biological fluid is passed through asecond layer of glass fiber to capture vesicles having a minimum sizefrom about 0.6 microns to about 0.8 microns in diameter, and having amaximum size of less than 1.6 microns. In several embodiments,combinations of glass-like and non-glass-like materials are used. In oneembodiment, a non-glass-like material comprising nitrocellulose is usedin combination with a glass-like material.

In several embodiments, the vesicle-capture material is modified inorder to tailor the profile of vesicles that are captured. In oneembodiment, the zeta potential of the material is used as a basis formodification (e.g., electrostatic charging) of the material. In severalembodiments, the material (based on its zeta potential) does not requiremodification.

In several embodiments, the methods disclosed herein further compriseeluting the vesicles from the vesicle-capture material. In someembodiments, the vesicle-capture material is optimized to balance theattractive nature of the vesicle-capture material and the ability of thevesicle-capture material to release captured vesicles. In someembodiments, vesicles are eluted from the vesicle-capture material bypassing a chaotropic reagent through vesicle-capture material. In someembodiments, vesicles are eluted from the vesicle-capture material bypassing a lysis buffer through vesicle-capture material.

In several embodiments, the vesicle-capture device is connected to avacuum source in order to pass the biological fluid from the sampleloading region through the vesicle-capture material and into the samplereceiving region. In one embodiment, the passings are accomplishedthrough the application of vacuum pressure to the device. In severalembodiments, the vesicle-capture device can receive positive pressure inorder to pass the biological fluid from the sample loading regionthrough the vesicle-capture material and into the sample receivingregion. In one embodiment, the passings are accomplished through theapplication of positive pressure to the device. In several embodiments,the device can be placed in a centrifuge in order to pass the biologicalfluid from the sample loading region through the vesicle-capturematerial and into the sample receiving region. In one embodiment, thepassings are accomplished through low-speed centrifugation of thedevice. In some embodiments, the passage of the biological fluid intothe sample receiving region is achieved by wicking-type materials. Inseveral embodiments, the vesicle capture device is configured in amulti-well plate format.

There is also provided herein a method for isolating a biomarker,comprising isolating vesicles comprising at least one biomarker from abiological fluid by passing the biological fluid through avesicle-capture material, removing non-vesicle material from thevesicle-capture material and lysing the vesicles in or on thevesicle-capture material with a lysis buffer, thereby isolating abiomarker from the vesicles.

In some embodiments, the biomarker is selected from the group consistingof RNA, DNA, protein, exosomes, vesicles, other circulating membranebound nucleic acid and/or protein-containing structures andcarbohydrate. In several embodiments, the RNA is of a type selected fromthe group consisting of mRNA, miRNA, rRNA, tRNA, and vRNA.

Some embodiments provide a device for the collection of vesicles from abiological fluid, the device comprising (1) at least one sample loadingregion; (2) at least one corresponding vesicle-capture material and (3)at least one corresponding sample receiving region, wherein passage ofthe biological fluid from the sample loading region through thevesicle-capture material and into the sample receiving region results incapture of vesicles within the biological fluid on or in thevesicle-capture material. In some embodiments, wherein thevesicle-capture material comprises glass-like materials, which have astructure that is disordered or “amorphous” at the atomic scale, likeplastic or glass. Glass-like materials include, but are not limited toglass beads or fibers, silica beads (or other configuration),nitrocellulose, nylon, polyvinylidene fluoride (PVDF) or other similarpolymers, metal or nano-metal fibers, polystyrene, ethylene vinylacetate or other co-polymers, natural fibers (e.g., silk), alginatefiber, or combinations thereof. In certain embodiments, thevesicle-capture material optionally comprises a plurality of layers ofvesicle-capture material. In other embodiments, the vesicle-capturematerial further comprises nitrocellulose. In some embodiments, thevesicle-capture material captures exosomes ranging in size from about 50to about 100 nanometers.

Some embodiments provide a method of isolating vesicles from biologicalfluid, comprising (1) obtaining a biological sample comprising vesicles;(2) loading at least a portion of the biological sample into a sampleloading region of a vesicle capture device; (3) passing the biologicalsample from the sample loading region through a vesicle-capture materialin the vesicle capture device; and (4) passing the biological samplefrom the vesicle-capture material to a sample receiving region, whereinthe passages of the biological sample results in capture of the vesicleswithin the biological fluid on or in the vesicle-capture material. Insome embodiments, the method further comprises eluting the vesicles fromthe vesicle-capture material. In some embodiments, the method furthercomprises capturing, enriching, and/or condensing vesicles comprisingRNA; removing non-vesicle material from the device; and lysing thevesicles in or on the vesicle-capture material with a lysis buffer,thereby isolating vesicle-associated RNA from the vesicles.

The methods summarized above and set forth in further detail belowdescribe certain actions taken by a practitioner; however, it should beunderstood that they can also include the instruction of those actionsby another party. Thus, actions such as “administering a blood test”include “instructing the administration of a blood test.”

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a cross-section view of one embodiment of a capture device asdisclosed herein.

FIG. 2 is a cross-section view of one embodiment of a first hollow bodyas disclosed herein.

FIG. 3 is a cross-section view of one embodiment of a second hollow bodyas disclosed herein.

FIG. 4 is a cross-section view of an additional embodiment of a secondhollow body as disclosed herein.

FIG. 5 is a cross-section view of microvesicle capture system asdisclosed herein.

FIG. 6 depicts data related to the exosome capture efficiency afteradjusting the salt and pH of urine samples

FIG. 7 depicts data related to the efficacy of exosome capture with thedevices disclosed herein as compared to ultracentrifugation-basedisolation.

FIGS. 8-1 and 8-2 depicts data related to the intra-assay variation whenthe devices disclosed herein are used to capture exosomes as compared toultracentrifugation-based isolation.

FIG. 9 depicts the comparison of an RNA profile detected after exosomecapture using the devices disclosed herein as compared toultracentrifugation-based isolation.

FIG. 10A depicts data related to the similar RNA profile detected from a12-hr urine sample when analyzed after capture of exosomes using thedevices disclosed herein as compared to ultracentrifugation-basedisolation.

FIG. 10B depicts data related to the consistency of RNA detection over atwo-week time period when exosomes recaptured using the devicesdisclosed herein.

DETAILED DESCRIPTION

General

Due to the rapid rate of nucleic acid degradation in the extracellularenvironment, conventional understanding suggests that many tissues areunable to provide nucleic acid that would be suitable as a diagnostictarget because the nucleic acids would be degraded before they could beused as a template for detection. However, extracellular RNA (as well asother biomarkers disclosed herein) is often associated with one or moredifferent types of membrane particles (ranging in size from 50-80 nm),exosomes (ranging in size from 50-100 nm), exosome-like vesicles(ranging in size from 20-50 nm), and microvesicles (ranging in size from100-1000 nm). Other vesicle types may also be captured, including, butnot limited to, nanovesicles, vesicles, dexosomes, blebs, prostasomes,microparticles, intralumenal vesicles, endosomal-like vesicles orexocytosed vehicles. As used herein, the terms “exosomes” and “vesicles”are used in accordance with their respective ordinary meanings in thisfield and shall also be read to include any shed membrane bound particlethat is derived from either the plasma membrane or an internal membrane.For clarity, the terms describing various types of vesicles shall,unless expressly stated otherwise, be generally referred to as vesiclesor exosomes. Exosomes can also include cell-derived structures boundedby a lipid bilayer membrane arising from both herniated evagination(e.g., blebbing) separation and sealing of portions of the plasmamembrane or from the export of any intracellular membrane-boundedvesicular structure containing various membrane-associated proteins oftumor origin, including surface-bound molecules derived from the hostcirculation that bind selectively to the tumor-derived proteins togetherwith molecules contained in the exosome lumen, including but not limitedto tumor-derived microRNAs or intracellular proteins. Exosomes can alsoinclude membrane fragments. Circulating tumor-derived exosomes (CTEs) asreferenced herein are exosomes that are shed into circulation or bodilyfluids from tumor cells. CTEs, as with cell-of-origin specific exosomes,typically have unique biomarkers that permit their isolation from bodilyfluids in a highly specific manner. As achieved by several embodimentsdisclosed herein, selective isolation of any of such type of vesiclesallows for isolation and analysis of their RNA (such as mRNA, microRNA,and siRNA) which can be useful in diagnosis or prognosis of numerousdiseases. Thus, exosomes and microvesicles (EMV) can provide biomarkersfor diseases (including, but not limited to, the isolation of vesiclesfrom urine for the assessment of renal disease). Target compounds thatcan be extracted using the devices and methods herein disclosed includeproteins, lipids, antibodies, vitamins, minerals, steroids, hormones,cholesterol, amino acids, vesicles, exosomes, and nucleic acids.

In several embodiments, biological fluid samples are processed. As usedherein, a “bodily fluid” shall be given its ordinary meaning and shallalso refer to a sample of fluid collected from the body of the subject,including but not limited to, for example, blood, plasma, serum, urine,sputum, spinal fluid, pleural fluid, nipple aspirates, lymph fluid,fluid of the respiratory, intestinal, and genitourinary tracts, tearfluid, saliva, breast milk, fluid from the lymphatic system, semen,cerebrospinal fluid, intra-organ system fluid, ascitic fluid, tumor cystfluid, amniotic fluid and combinations thereof.

In several embodiments, a biological fluid sample is processed by usinga system configured to capture a target of interest from the fluid.Generally speaking several embodiments of the system comprise a firstfluid compartment having a first volume, the first compartment beingconfigured to reversibly interconnect to a second fluid compartmentcomprising an agent capture material, with both the first and secondcompartment being dimensioned to fit within an outer housing. In severalembodiments, the outer housing functions to receive eluate (e.g., thebiological sample depleted of the target of interest).

In several embodiments the system is particularly advantageous in thatit allows a high degree of concentration of agents of interest that arepresent at low concentrations in a fluid sample.

One aspect of the present disclosure relates to devices and methods forthe enhanced efficiency of capturing exosomes, vesicles, and othercirculating membrane bound nucleic acid and/or protein-containingstructures from biological fluids. These devices and methods canadvantageously be employed even when the individual samples have variedcharacteristics. The captured exosomes, vesicles and other circulatingmembrane bound nucleic acid and/or protein-containing structures maycontain a variety of different specific biomarkers, which can beemployed in a variety of diagnostic, prognostic and therapeutic andother medically-related methods and uses.

FIG. 1 depicts an embodiment of a capture device 100. The embodiment ofcapture device 100 depicted in FIG. 1 comprises a first hollow body 1 infunctional communication with a second hollow body 2. “Functionalcommunication” shall be given its ordinary meaning and shall also referto the two hollow bodies being coupled in such a manner that it ispossible to carry out an intended use of the device. Direct and indirectconnections are within the scope of the meaning of “functionalcommunication.”

In several embodiments, a fluid sample 3 is loaded into first hollowbody 1 and passed to second hollow body 2. In several embodiments, fluidsample 3 passes through a capture material 4. Fluid sample 3 may containa target component. In some embodiments, as fluid sample 3 passesthrough capture material 4, capture material 4 retains at least some ofthe target component contained in fluid sample 3. In some embodiments, atarget component comprises at least one exosome though other componentswhose isolation or purification is desirable may also be considered astarget components.

In some embodiments, capture material 4 is located within second hollowbody 2. In several embodiments, after fluid sample 3 has passed throughcapture material 4, second hollow body 2 is removed from first hollowbody 1, and second hollow body 2 is then processed to retrieve thetarget components retained in capture material 4. In at least oneembodiment, exosomes that have been retained by capture material 4 aresubsequently recovered from capture material 4 by passing a small amountof liquid (e.g., a lysis buffer) through capture material 4. In someembodiments, another solution (e.g., a washing buffer) is optionallypassed through capture material 4 before and/or after application of theliquid used to recover the retained exosomes.

In some embodiments, gravitational force drives the passage of fluidsample 3 through capture material 4. In some embodiments, a positivepressure drives fluid sample 3 through capture material 4. In someembodiments, a negative pressure drives fluid sample 3 through capturematerial 4. In several embodiments, no negative or positive pressure isused. In some embodiments, centrifugal force drives fluid sample 3through capture material 4. In some embodiments, a wicking-type materialdrives fluid sample 3 through capture material 4. In some embodiments,capillary action drives fluid sample 3 through capture material 4.

Fluid sample 3 can be any liquid including bodily fluids. “Bodily fluid”shall be given its ordinary meaning and shall also refer to a sample offluid isolated from anywhere in the body of the subject, including butnot limited to, for example, blood, plasma, serum, urine, sputum, spinalfluid, pleural fluid, nipple aspirates, lymph fluid, fluid of therespiratory, intestinal, and genitourinary tracts, tear fluid, saliva,breast milk, fluid from the lymphatic system, semen, cerebrospinalfluid, intra-organ system fluid, ascitic fluid, tumor cyst fluid,amniotic fluid, and combinations thereof.

FIG. 2 depicts one embodiment of first hollow body 1. In severalembodiments, first hollow body 1 has an inlet opening 101, an outletopening 102, an outer surface 130, and an inner surface 140. In someembodiments, inlet opening 101 is a circular opening having an inletdiameter 111. In some embodiments, outlet opening 102 is a circularopening having an outlet diameter 112. In several embodiments, inletopening 101 and outlet opening 102 are circular openings that areaxially-aligned, with outlet diameter 112 being smaller than inletdiameter 111.

In some embodiments, first hollow body 1 comprises an upper region 132,an intermediate region 134, and a terminal region 136. In someembodiments, upper region 132 and terminal region 136 are cylindrical orsubstantially cylindrical, and intermediate region 134 is tapered (e.g.,conical). In some embodiments, the taper of intermediate region 134 isconfigured to facilitate passage of fluid sample 3 through outletopening 102. In some embodiments, first hollow body 1 includes a collar105 that extends beyond outer surface 130 of an adjacent portion offirst hollow body 1. In some embodiments, collar 105 is configured tosupport first hollow body 1 when first hollow body 1 is inserted into astorage rack or a receiving vessel (not shown).

FIG. 3 depicts an embodiment of second hollow body 2. In severalembodiments, second hollow body 2 has an inlet opening 201, an outletopening 202, an outer surface 230, and an inner surface 240. In someembodiments, inlet opening 201 is a circular opening having an inletdiameter 211. In some embodiments, outlet opening 202 is a circularopening having an outlet diameter 212. In several embodiments, inletopening 201 and outlet opening 202 are circular openings that areaxially-aligned, with outlet diameter 212 being smaller than inletdiameter 211.

In several embodiments, first hollow body 1 and second hollow body 2 aremade of material that has a low binding affinity for nucleic acids.Suitable materials include, but are not limited to, plastics such aspolypropylene, polystyrene, and polyethylene, among others. In someembodiments, first hollow body 1 and second hollow body 2 are made ofmetal or composite material. In some embodiments, inner surfaces 140,240 are coated with one or more substances that lowers the bindingaffinity of the surfaces for nucleic acids.

In some embodiments, second hollow body 2 comprises an upper region 232,an intermediate region 234, and a terminal region 236. In someembodiments, terminal region 236 is tapered. In at least one embodiment,the taper of terminal region 236 is configured to facilitate passage offluid sample 3 out of second hollow body 2.

In several embodiments, second hollow body 2 has a tab 260 that extendsfrom outer surface 230. In some embodiments, tab 260 is located in upperregion 232. Tab 260 has an upper surface 262. In some embodiments, uppersurface 262 is substantially co-planar with inlet opening 201. Inseveral embodiments, upper surface 262 is sufficiently dimensioned toserve as a platform for labeling second hollow body 2. In at least oneembodiment, upper surface 262 is between about 1 mm to about 5 mm wideand about 1 mm to about 5 mm long. In some embodiments, a label 264 isaffixed to upper surface 262. In several embodiments, upper surface 262is marked by any suitable means including ink, or etching. In at leastone embodiment, label 264 or the marking of upper surface 262 denotesthe identity (e.g., the source patient) of the fluid sample 3 that hasbeen passed through second hollow body 2. In some embodiments, label 264or marking of upper surface 262 encodes a bar code (e.g., a 2D or 3D barcode). In several embodiments, RFID tags or other identifiers may beused to denote the patient identity from which the sample was obtained.

In several embodiments, upper region 232 of second hollow body 2 isconfigured to functionally communicate with terminal region 136 of firsthollow body 1. First hollow body 1 and second body 2 may functionallycommunicate by any number of ways including but not limited to matingscrew threads, an interference fit, and a compression fitting. In someembodiments, terminal region 136 of first hollow body 1 is configured tofit inside upper region 232 of second hollow body 2. In someembodiments, upper region 232 of second hollow body 2 is configured tofit inside terminal region 136 of first hollow body 1. In someembodiments, at least a portion of outer surface 130 is surrounded by atleast a portion of inner surface 240. In some embodiments, at least aportion of outer surface 230 is surrounded by at least a portion ofinner surface 140. In some embodiments, outlet diameter 112 is smallerthan inlet diameter 211.

In some embodiments, first hollow body 1 has at least one pin 150 thatprotrudes from outer surface 130 of terminal region 136, and secondhollow body 2 has at least one channel 250 in upper region 232 of secondhollow body 2 (see e.g., FIG. 3). In at least one embodiment, pin 150 isconfigured to reversibly cooperate with channel 250. Channel 250 has alongitudinal portion 252, a transverse portion 254, and a retrogradeportion 256. In some embodiments, first hollow body 1 is coupled tosecond hollow body 2 by sliding pin 150 into longitudinal portion 252 ofchannel 250. First hollow body 1 and second hollow body 2 are positionedto allow pin 150 to reach transverse portion 254 of channel 250. Secondhollow body 2 is then rotated to bring pin 150 into transverse portion254 until pin 150 lines up with retrograde portion 256 of channel 250.The compressive force between first hollow body 1 and second hollow body2 is then reduced, allowing pin 150 to slide into retrograde portion256, thereby securing a coupling between first hollow body 1 and secondhollow body 2. In some embodiments, second hollow body 2 is removed fromfirst hollow body 1 by squeezing the two hollow bodies together andallowing pin 150 to retrace channel 250.

In some embodiments, the at least one channel 250 in upper region 232 ofsecond hollow body 2 comprises a longitudinal portion 252 and atransverse portion 254. In some embodiments, first hollow body 1 iscoupled to second hollow body 2 by sliding pin 150 into longitudinalportion 252 of channel 250. First hollow body 1 and second hollow body 2are positioned to allow pin 150 to reach transverse portion 254 ofchannel 250. Second hollow body 2 is then rotated to bring pin 150 intotransverse portion 254 thereby securing a coupling between first hollowbody 1 and second hollow body 2. After processing, second hollow body 2is removed from first hollow body 1 by rotating the two hollow bodies inthe opposite direction and allowing pin 150 to retrace channel 250,thereby allowing the first and second hollow bodies to disengage. Inseveral embodiments, the first and second hollow body are configured toreversible interact in a manner that allows their decoupling to occurwithout requiring second hollow body to be re-passed through theinterior of first hollow body. That is, certain devices may allowinteraction of the first and second bodies by virtue of the secondhollow body being inserted into, and partially through, the first hollowbody (e.g., the second body nests inside the first body). While thisprovides some advantages (e.g., security of the interaction duringcentrifugation or other handling) this then requires the second hollowbody to retrace its path (e.g., an upward path) through the first hollowbody to disengage the two. This presents a potential issue with respectto cross contamination. Advantageously, in several embodiments, thesecond hollow body of the devices disclosed herein can be de-coupledfrom the first hollow body without requiring the second hollow body topass through the first hollow body. In several embodiments, this greatlyreduces the risk for contamination of the second hollow body, but thereversible interaction between the two bodies is sufficient to maintaina secure interaction during centrifugation (or other handling).

In several embodiments, capture material 4 is made from any suitablematerial that can retain the target component being extracted from fluidsample 3. In several embodiments, the material used for capture material4 is optimized to balance the attractive nature of the material for thetarget component and the ability of the material to release the targetcomponent under appropriate conditions.

In some embodiments, capture material 4 is optionally modified to tailorthe profile of target components retained by capture material 4. In someembodiments, capture material 4 is electrocharged (e.g.,electrostatically charged), coated with hydrophilic or hydrophobicmaterials, chemically modified, and/or biologically modified. In severalembodiments, the zeta potential of capture material 4 is used as a basisfor modification (e.g., electrostatic charging) of the material. In someembodiments, capture material 4 (based on its zeta potential) does notrequire modification. In some embodiments, capture material 4 ismodified by attaching a nucleotide sequence to the surface of capturematerial 4. In some embodiments, a protein is attached to the surface ofcapture material 4. In some embodiments, biotin or streptavidin isattached to the surface of capture material 4. In some embodiments, anantibody or antibody fragment is attached to capture material 4. Any ofsuch embodiments can be employed to advantageously increase theefficiency of capture of a target.

In some embodiments, differential capture of vesicles is achieved basedon the surface expression of protein markers and a complementary agenton capture material 4 which identifies that marker (e.g., an antibodythat recognizes an antigen on a particular vesicle). In someembodiments, the markers are unique vesicle proteins or peptides. Insome disease states, the markers may also comprise certain vesiclemodifications, which, in some embodiments, are used to isolateparticular vesicles. In such embodiments, capture material 4 may beconfigured in a manner which allows for specific recognition of thevesicle modification. Modification of the vesicles may include, but arenot limited to the addition of lipids, carbohydrates, and othermolecules such as acylated, formylated, lipoylated, myristolylated,palmitoylated, alkylated, methylated, isoprenylated, prenylated,amidated, glycosylated, hydroxylated, iodinated, adenylated,phosphorylated, sulfated, and selenoylated, ubiquitinated. In someembodiments, capture material 4 is configured to recognize vesiclemarkers comprising non-proteins such as lipids, carbohydrates, nucleicacids, RNA, mRNA, siRNA, microRNA, DNA, etc.

In some embodiments, the interactions between vesicles and capturematerial 4 are based on electrostatic interaction, hydrophobicinteraction, van der Waals force, or a combination of theseinteractions. Thus, the biochemical makeup of the sample comprising thevesicles can alter these forces, possibly to a degree that significantlyhampers the capture efficiency.

In several embodiments, a target range for capture conditions that thevesicles are exposed to when passed over/through the capture materialscomprise between about 1 mM and about 1000 mM monovalent cation (e.g.,sodium and/or potassium), including ranges having a lower concentrationof about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM,about 60 mM, about 70 mM, about 80 mM, about 90 mM or about 100 mm,about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM,about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM(and any concentration therebetween) and upper concentrations of about500 mM, about 600 mM, about 700 mM, about 800 mM, and about 900 mM (andany concentration therebetween). Thus, in several embodiments theconcentration ranges are from about 20 mM to about 900 mM, from about 20mM to about 800 mM, from about 30 mM to about 700 mM, and from about 40mM to about 600 mM, and overlapping ranges thereof. In conjunction withthose conditions, the pH is adjusted, in several embodiments, from about4, about 5, or about 6 to about 9 or about 10 (or pH values betweenthose listed). Thus, depending on the embodiment, pH ranges include fromabout 4 to about 10, from about 5 to about 9, and from about 6 to about9.

In some embodiments, the materials used for capture material 4 comprisematerials that inhibit the capture of vesicles. Thus, in severalembodiments, capture material 4 is pre-treated to remove such inhibitorymaterials in advance of using the capture material to capture thevesicles. For example, high concentrations of proteins such as albuminmay lower the capture efficiency of vesicle capture. In such cases,albumin can be removed by various techniques, such as, for example,passing materials or solutions through or over capture material 4, thematerials or solutions comprising a compound (e.g., Blue Trisacryl Mresin) with a greater affinity for the albumin than the albumin has forcapture material 4. The techniques used to remove contaminants may alsoinclude heating, acid bath, basic bath, ultrasonic cleaning, and thelike.

In several embodiments, it is advantageous to adjust the biochemicalcharacteristics of fluid sample 3 to preferred ranges (e.g., saltconcentration, pH, etc.) prior to attempting to capture the vesicles. Inseveral embodiments, a buffer solution such as phosphate buffer saline(PBS) or HEPES buffer is used. In several embodiments, the pH of suchbuffers ranges from a pH of about 6 to about 9. In several embodiments,the concentration of monovalent cations such as sodium and potassium isgreater than about 50 mM, greater than about 60 mM, greater than about70 mM, greater than about 80 mM, greater than about 90 mM, greater thanabout 100 mM, greater than about 200 mM, and sometimes may require evengreater concentrations, depending on the embodiment. In severalembodiments, the end result of the mixture of the urine and buffersolution is between about 20 mM and about 600 mM monovalent cation, suchas sodium and potassium, and between about pH 6 and about pH 9.Capturing vesicles can then be performed as discussed in more detailbelow, and analysis performed as described below.

In several embodiments, capture material 4 is made of glass-likematerial. In some embodiments, capture device 100 includes a filtermaterial 5 (shown in FIG. 2) that is configured to filter fluid sample 3before fluid sample 3 passes through capture material 4. In someembodiments filter material 5 is placed in second hollow body 2 betweencapture material 4 and inlet opening 201. In some embodiments, filtermaterial 5 is placed in first hollow body 1 between intermediate region136 and outlet opening 102. In several embodiments, however, no filtermaterial is used.

In several embodiments, combinations of filter material 5 and capturematerial 4 are used. In some embodiments, capture material 4 comprises aplurality of layers of material. In several embodiments, capturematerial 4 comprises at least a first layer and a second layer of glassfiber. In some embodiments, fluid sample 3 is passed through filtermaterial 5 to capture components that are about 1.6 microns or greaterin diameter. In some embodiments, fluid sample 3 is passed throughcapture material 4 so as to capture vesicles having a minimum size fromabout 0.6 microns to about 0.8 microns in diameter, and having a maximumsize of less than about 1.6 microns. In several embodiments, theretention rate of capture material 4 is greater than about 50%, about75%, about 90%, or about 99% for vesicles having a diameter of fromabout 0.6 microns to about 1.5 microns in diameter. In at least oneembodiment, capture material 4 captures vesicles sized from about 0.7microns to about 1.6 microns in diameter. In at least one embodiment,capture material 4 captures exosomes or other vesicles ranging in sizefrom about 0.020 microns to about 1.0 microns.

In several embodiments, capture material 4 comprises combinations ofglass-like and non-glass-like materials. For example, in one embodiment,a non-glass-like material comprising nitrocellulose is used. In someembodiments, capture material 4 comprises glass-like materials, whichhave a structure that is disordered, or “amorphous” at the atomic scale,such as plastic or glass. Glass-like materials include, but are notlimited to, glass beads or fibers, silica beads (or otherconfigurations), nitrocellulose, nylon, polyvinylidene fluoride (PVDF)or other similar polymers, metal or nano-metal fibers, polystyrene,ethylene vinyl acetate or other co-polymers, natural fibers (e.g.,silk), alginate fiber, or combinations thereof. Other suitable materialsfor capture material 4 include zeolite, metal oxides or mixed metaloxides, aluminum oxide, hafnium oxide, zirconium oxide, or combinationsthereof.

In some embodiments, vesicles are retained in capture material 4 byvirtue of the vesicle having physical dimensions that prohibit thevesicle from passing through the spaces of capture material 4 (e.g.,physical retention based on size). In some embodiments, vesicles areretained in capture material 4 by bonding forces between the vesicle andcapture material 4. In some embodiments, vesicles form antigen-antibodybonds with capture material 4. In several embodiments, vesicles formhydrogen bonds with capture material 4. In some embodiments, van derWaals forces form between the vesicle and capture material 4. In someembodiments, nucleotide sequences of the vesicle bind to nucleotidesequences attached to capture material 4.

In several embodiments, capture device 100 is used in conjunction with areceiving vessel 500 (see FIG. 5) that receives fluid sample 3 in areceiving compartment 600 after fluid sample 3 has passed throughcapture device 100. In some embodiments, the receiving vessel alsoincludes a cap 700, to secure the capture device 100 within thereceiving vessel 500 during processing. In several embodiments, the capis a press-fit cap, while in other embodiments the cap comprises ascrew-fit cap. In several embodiments, the receiving vessel comprises acentrifuge tube, thus, in some embodiments, first hollow body 1 andsecond hollow body 2 are sized to fit within a receivingvessel/centrifuge tube. In some embodiments, collar 105 serves as ameans for holding capture device 100 in a fixed position relative to thereceiving vessel. In several embodiments, capture device 100 and collar105 are sized to permit use of capture device 100 with a receivingvessel such as a 10 mL, 12 mL, 15 mL, 30 mL, 50 mL, 175 mL, or 225 mLcentrifuge tube, though centrifuge tubes of other sizes and capacitiesare also contemplated. In some such embodiments, collar 105 is sized tofit over the mouth of the centrifuge tube without obstructing thefunction of the threaded cap of the centrifuge tube. In severalembodiments, capture device 100 is placed within a centrifuge tube, andcentrifugal force is applied to drive fluid sample 3 from first hollowbody 1 through capture material 4 and into second hollow body 2.

In some embodiments, capture device 100 is sized so that outlet opening202 of second hollow body 2 does not contact fluid sample 3 after fluidsample 3 has passed through capture device 100 and accumulated in thereceiving vessel. In some embodiments, the volume capacity of thereceiving vessel is greater than the volume capacity of capture device100 by about 2-fold, by about 3-fold, by about 4-fold, or by about5-fold.

In some embodiments, capture device 100 has a volume sufficient toreceive the entire fluid sample 3 and other reagents to facilitatebinding of nucleic acids to capture material 4. In some embodiments,capture device 100 is sized to accommodate a volume of between about 1mL and 1000 mL, including between about 1 mL and 100 mL, between about 5mL and 50 mL, between about 10 mL and 20 mL, and any volumes betweenthose ranges. In some embodiments, capture device 100 accommodates avolume of about 15 mL.

In some embodiments, the capacity of first hollow body 1 is greater thanthe capacity of second body 2 by about 100-fold, or by about 50-fold, orby about 20-fold, or by about 10-fold, or by about 5-fold. In someembodiments, the capacity of first hollow body 1 is about the same asthe capacity of second hollow body 2.

In many embodiments, the dimensions of capture material 4 are optimizedto balance having sufficient capture material 4 to adequately capture atarget from sample 3 while also allowing a small volume of liquid (e.g.,microliter scale) to be used to elute the bound target components.Reducing the volume of recovery liquid allows, in certain advantageousembodiments, target components to be extracted at higher concentrations.In some embodiments, the volume of capture device 100 is greater thanthe volume of capture material 4 by about 1000-fold, by about 500-fold,by about 300-fold, or by about 100-fold. In embodiments where thematerial of capture material 4 includes interstitial spaces, the meaningof the phrase “volume of capture material 4” shall be taken to includethe volume of these interstitial spaces. In several embodiments, theelution volume ranges from about 5 to about 500 microliters, includingabout 5 microliters to about 10 microliters, about 10 microliters toabout 20 microliters, about 20 microliters to about 50 microliters,about 50 microliters to about 100 microliters, about 100 microliters toabout 150 microliters, about 150 microliters to about 200 microliters,about 200 microliters to about 300 microliters, about 300 microliters toabout 400 microliters, about 400 microliters to about 500 microliters,and overlapping ranges therebetween.

In some embodiments, capture material 4 is cuboidal. In some embodimentscapture material 4 is wafer-shaped, spherical, or some combinationthereof. In some embodiments capture material 4 has a surface area tothickness ratio of about 50:1, about 25:1, about 10:1, about 5:1, orabout 3:1. In some embodiments, capture material 4 is a cylindricalwafer having a diameter to length ration of about 20:1, about 10:1,about 5:1, or about 2:1. In at least one embodiment, capture material 4is cylindrical and has a diameter of about 9 mm and a thickness of about1 mm.

In some embodiments, a fluid sample is passed through the device by wayof application of positive pressure. For example, in some embodiments,the first hollow body 1 is configured to receive a syringe plunger,which, when depressed toward second hollow body, provides a positivepressure that drives fluid sample 3 through capture device 100. In someembodiments, a fluid sample is passed through the device by way ofapplication of negative pressure. For example, in some embodiments, thesecond hollow body is adapted to reversibly connect to a vacuum source,such as a vacuum manifold, thereby allowing application of a negativepressure that drives fluid sample 3 through capture device 100. In someembodiments employing a receiving vessel, the receiving vessel isconfigured to pass a negative (or positive, depending on the embodiment)pressure to the capture device, thereby allowing the fluid sample to bepassed through the capture device. However, in several embodiments, nospecific positive or negative pressure is applied. For example, inseveral embodiments, centrifugal forces are applied to drive fluidsample 3 through capture device 100. Gravitational flow may also beused, in several embodiments.

In some embodiments, terminal region 236 of second hollow body 2 issized to fit within a well of a standard multi-well plate. In severalembodiments, terminal region 236 is sized to fit within a well of astandard 6-well plate, or a standard 12-well plate, or a standard24-well plate, or a standard 96-well plate, or a standard 384-wellplate, or a standard 1536-well plate, etc. Such plates are commerciallyavailable from various manufacturers, including but not limited to,Corning, Nunc, Fisher, BD Biosciences, etc. In several embodiments, theplates have well dimensions that are shown in Table 1.

TABLE 1 Example Microplate Dimensions for Use with Capture SystemsNumber Well Diameter of Wells Plate Length (mm) Plate Width (mm) (mm, attop of well) 6 127.76 85.47 35.43 12 127.89 85.6 22.73 24 127.89 85.616.26 48 127.89 85.6 11.56 96 127.8 85.5 6.86

In some embodiments, tab 260 of second hollow body 2 extends over atleast a portion of a neighboring well of a multi-well plate when secondhollow body 2 interacts with a first well of the multi-well plate. In atleast one embodiment, tab 260 is configured to allow half of the wellsof a multi-well plate to be occupied at a time by second hollow bodies 2without tabs 260 overlapping with one another. In some embodiments,second hollow body 2 has a protrusion 270 that interacts with a wall ofa well of a multi-well plate and secures second hollow body 2 to a wellof the multi-well plate. In several embodiments, tab 260 is dimensionedso that each well of a multi-well plate can be used to receive a sample.

In several embodiments, a method for isolating a biomarker comprisestaking a fluid sample 3 from a patient, passing the fluid sample 3through capture material 4, removing non-vesicle material from capturematerial 4, and lysing the vesicles in or on capture material 4 with alysis buffer, thereby isolating a biomarker from the vesicles. In someembodiments, the biomarker is selected from the group consisting of RNA,DNA, protein, and carbohydrate. In several embodiments, the RNA is of atype selected from the group consisting of mRNA, miRNA, rRNA, tRNA, andvRNA.

In some embodiments, capture device 100 is placed within a centrifugetube, and collar 105 holds capture device 100 in a fixed positionrelative to the centrifuge tube. Fluid sample 3 is loaded into capturedevice 100 before or after placing capture device 100 within thecentrifuge tube. Capture device 100 is subjected to centrifugation. Thecentrifuge tube serves as a receiving vessel and receives fluid sample 3after it has passed through capture device 100. In some embodiments,low-speed centrifugation is used to drive fluid sample 3 through capturedevice 100.

In some embodiments, a kit is provided for extracting target componentsfrom fluid sample 3. Kits often allow better management of qualitycontrol and better consistency in results. In some embodiments, a kitcomprises a capture device 100 and additional items useful to carry outmethods disclosed herein. In some embodiments, a kit comprises reagentsselected from the group consisting of lysis buffers, chaotropicreagents, washing buffers, alcohol, detergent, or combinations thereof.In some embodiments, kit reagents are provided individually or instorage containers. In several embodiments, kit reagents are providedready-to-use. In some embodiments, kit reagents are provided in the formof stock solutions that are diluted before use. In some embodiments, akit comprises plastic parts that are useful to carry out methods hereindisclosed. In some embodiments, a kit comprises plastic parts selectedfrom the group consisting of racks, centrifuge tubes, vacuum manifolds,and multi-well plates. Instructions for use are also provided, inseveral embodiments.

EXAMPLES Example 1—Effect of pH/Salt Concentration on Exosome Capture

The impacts of various characteristics of a biological sample wereevaluated with respect to the efficacy of exosome capture. Urine samplescollected from four healthy donors were centrifuged at 800×g for 15 minand the supernatants were collected. 4.5 mL of urine supernatant fromeach subject was mixed with different volumes of concentrated buffersolution prior to processing. Samples were processed by the collectiondevice disclosed herein. In brief, the samples were added to the firstbody, which was connected to the second body. The first and secondbodies were placed inside a 50 mL conical centrifuge tube (receivingvessel) and centrifuged at 2,000×g 10 min to capture exosomes andmicrovesicles (EMV) on a capture filter within the second body.Thereafter, the first and second bodies were removed from the receivingvessel and the second body was disengaged from the first body. Thesecond body was placed with its outlet portion in a well of a multiwellmicroplate. After lysing the captured EMV by a lysis buffer (37° C. 10min), the lysates were transferred to an oligo(dT) immobilizedmicroplate (Hitachi Chemical Research Center, Inc.) for mRNA isolation.Several kidney-related genes including housekeeping mRNAs werequantified by real-time RT-PCR. For protocol comparison, a standardultracentrifugation protocol was used for exosome isolation. The 800×gsupernatants were centrifuged at 100,000×g for 1 hour and the EMVpellets were collected. After lysing the EMV by a lysis buffer, thelysates were transferred to an oligo(dT) immobilized microplate andprocessed for mRNA isolation and real-time RT-PCR.

As shown in FIG. 6 addition of a buffer solution to adjust the pH andthe salt concentration of urine sample improve the efficiency of exosomecapture using the device is disclosed herein. FIG. 6, as depicted by thearrows, improve the assay sensitivity of urine samples collected fromsubject #1 (based on expression of GAPDH and RPLPO housekeeping genes)but do not adversely impact sensitivity of the samples from othersubjects.

Example 2—Filter Based Exosome Capture Compared to StandardUltracentrifugation

Many commonly used protocols and play ultracentrifugation to captureexosome from biological fluids. However, as discussed above,ultracentrifugation can be cost intensive. On exosome capture device, asdisclosed herein, was used to process 0.1 to 10 mL urine samples. Urinesamples were also processed to capture exosomes using establishedultracentrifugation methods. Identical mRNA isolation and PCR protocolswere then used.

FIG. 7 shows the results of PCR application of three housekeeping genes(beta actin, GAPDH and RPLPO) based on isolation using the disclosedexosome capture device (open circles) or ultracentrifugation of (opensquares). A high degree of correlation was detected between bothmethods, thereby indicating that the exosome capture devices disclosedherein are effective at capturing exosome and maintaining the mRNAwithin those exosomes.

FIG. 8 shows additional data related to the intra-assay variation whenexosome capture using the devices disclosed herein was compared withultracentrifugation-based methods. As shown in FIG. 8 and Table 2,highly similar mRNA expression profiles were detected using eithermethod, thus indicating that exosome capture using the devices disclosedherein provides reproducible and accurate mRNA results. Moreover, thesedata show a significantly reduced intra-assay variation when using theexosome capture devices disclosed herein. As such gene expressionanalysis using these devices can achieve higher degrees of accuracy andreduce the risk of false positive results based on data variability.

TABLE 2 Intra-assay Variation Using Exosome Capture Devices Std. GeneMethod Run 1 Run 2 Run 3 Run 4 Run 5 Run 6 Run 7 Run 8 Avg. Dev CV (%)Beta-Actin Device 26.2 26.3 25.9 25.9 26.4 25.9 25.9 25.9 26.0 0.2 0.9Ultra 27.3 24.9 27.0 24.9 26.4 27.0 25.5 27.9 26.4 1.1 4.3 GAPDH Device23.9 24.4 24.2 23.7 24.3 24.1 24.3 23.9 24.1 0.2 1.0 Ultra 25.5 23.125.5 22.7 24.2 25.2 23.7 25.2 24.4 1.1 4.6 RPLP0 Device 23.4 23.6 23.623.4 23.5 23.4 23.9 23.4 23.5 0.2 0.8 Ultra 24.9 22.3 25.0 22.1 23.424.1 22.9 24.7 23.7 1.2 4.9 PDCN Device 30.3 32.0 33.3 31.9 33.4 20.632.4 31.6 31.9 1.1 3.5 Ultra 33.9 31.2 40.0 31.8 32.5 40.0 33.2 40.035.3 4.0 11.2 SLC12A1 Device 27.1 27.7 26.4 26.9 27.1 26.8 27.6 26.927.1 0.4 1.5 Ultra 29.8 26.5 28.6 26.4 27.5 29.2 27.5 29.6 28.1 1.4 4.8ALB Device 25.9 26.2 24.9 25.5 25.9 25.7 26.2 25.5 25.7 0.4 1.7 Ultra28.9 25.9 30.6 25.4 27.1 28.5 27.2 28.3 27.7 1.7 6.1 Uromodulin Device29.3 29.5 28.8 29.5 28.6 28.7 29.6 29.6 29.2 0.4 1.4 Ultra 30.8 28.433.1 29.1 30.2 30.6 30.9 31.6 30.6 1.5 4.8 AQP2 Device 30.4 31.7 29.829.3 31.5 29.7 29.1 29.9 29.9 1.0 3.2 Ultra 40.0 30.1 32.5 29.0 30.640.0 29.9 40.0 40.0 5.1 14.9

An additional experiment was performed to determine the similarity inmRNA profile when exosomes were captured using the devices disclosedherein or using ultra centrifugation based protocols. Certain kidneyrelated genes, as well as a variety of housekeeping genes were amplifiedfrom urine samples (10 mL) processed through the devices disclosedherein or by ultracentrifugation. As shown in FIG. 9 very similarexpression profiles resulted, regardless of the method employed. Thesedata, in conjunction with the data above relating to reduced intra-assayvariability indicate that the devices disclosed herein can result inhighly accurate mRNA expression data.

Example 3—Urine Exosome Monitoring

The exosome capture devices disclosed herein are used to assess geneexpression in urine samples (12 hour collection) collected from asubject four times over a two-week period. FIG. 10A depicts this geneexpression data, and, notably, the level of gene expression (within eachgene tested) is highly similar throughout the two-week experimentalperiod. FIG. 10B depicts the data for three housekeeping genes overtime. Of note, is a highly stable gene expression profile of each ofthese genes, thereby confirming accuracy of mRNA expression profilingwhen exosomes are captured using the devices disclosed herein.

It is contemplated that various combinations or subcombinations of thespecific features and aspects of the embodiments disclosed above may bemade and still fall within one or more of the inventions. Further, thedisclosure herein of any particular feature, aspect, method, property,characteristic, quality, attribute, element, or the like in connectionwith an embodiment can be used in all other embodiments set forthherein. Accordingly, it should be understood that various features andaspects of the disclosed embodiments can be combined with or substitutedfor one another in order to form varying modes of the disclosedinventions. Thus, it is intended that the scope of the presentinventions herein disclosed should not be limited by the particulardisclosed embodiments described above. Moreover, while the invention issusceptible to various modifications, and alternative forms, specificexamples thereof have been shown in the drawings and are hereindescribed in detail. It should be understood, however, that theinvention is not to be limited to the particular forms or methodsdisclosed, but to the contrary, the invention is to cover allmodifications, equivalents, and alternatives falling within the spiritand scope of the various embodiments described and the appended claims.Any methods disclosed herein need not be performed in the order recited.The methods disclosed herein include certain actions taken by apractitioner; however, they can also include any third-party instructionof those actions, either expressly or by implication. For example,actions such as “administering a blood test” include “instructing theadministration of a blood test.” The ranges disclosed herein alsoencompass any and all overlap, sub-ranges, and combinations thereof.Language such as “up to,” “at least,” “greater than,” “less than,”“between,” and the like includes the number recited. Numbers preceded bya term such as “about” or “approximately” include the recited numbers.For example, “about 3 mm” includes “3 mm.”

What is claimed is:
 1. A method of isolating vesicles from biologicalfluid, the method comprising: (a) obtaining a urine sample comprisingsaid vesicles; (b) titrating said urine sample comprising said vesicleswith a buffer solution comprising either phosphate buffered saline orHEPES to adjust the salt concentration of the urine sample comprisingsaid vesicles to between 200 mM and 900 mM; (c) adjusting the pH of theurine sample comprising said vesicles to between about pH 6 and pH 9;and (d) passing said urine sample comprising said vesicles through avesicle-capture material, said vesicle-capture material comprisingglass-like materials, to produce a supernatant, wherein the vesiclesfrom said urine sample are captured on or in said vesicle-capturematerial, wherein said titrating and said adjusting the pH of the urinesample comprising said vesicles is performed prior to passing said urinesample comprising said vesicles through the vesicle-capture material. 2.The method of claim 1, wherein the supernatant is discarded.
 3. Themethod of claim 1, further comprising re-passing the supernatant throughthe vesicle-capture material.
 4. The method of claim 1, wherein saidsalt concentration is between 200 mM and 600 mM.
 5. The method of claim1, wherein the salt concentration is based on the concentration ofmonovalent cations in the sample.
 6. The method of claim 1, wherein thesalt concentration that is adjusted is the concentration of monovalentcations.
 7. The method of claim 1, wherein the said glass-like materialcomprises at least a first layer and a second layer of glass fiber. 8.The method of claim 7, wherein the said glass-like material isconfigured to have a greater than 50% retention rate for particleshaving a diameter of from 0.6 microns to 1.5 microns in diameter.
 9. Themethod of claim 1, wherein the vesicles are selected from the groupconsisting of exosomes, vesicles, and other circulating membrane boundnucleic acid and/or protein-containing structures.
 10. The method ofclaim 1, wherein the vesicles contain a biomarker selected from thegroup consisting of mRNA, miRNA, rRNA, tRNA, and vRNA.
 11. The method ofclaim 1, wherein the salt concentration of the urine sample is adjustedto between about 200 mM and 600 mM.
 12. The method of claim 1, whereinsaid adjusting the pH of the urine sample is performed prior to passingsaid urine sample through the vesicle-capture material.
 13. The methodof claim 12, wherein the salt concentration of the urine sample isadjusted to between about 200 mM and 600 mM.